Flavin-enabled reductive and oxidative epoxide ring opening reactions

Epoxide ring opening reactions are common and important in both biological processes and synthetic applications and can be catalyzed in a non-redox manner by epoxide hydrolases or reductively by oxidoreductases. Here we report that fluostatins (FSTs), a family of atypical angucyclines with a benzofluorene core, can undergo nonenzyme-catalyzed epoxide ring opening reactions in the presence of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH). The 2,3-epoxide ring in FST C is shown to open reductively via a putative enol intermediate, or oxidatively via a peroxylated intermediate with molecular oxygen as the oxidant. These reactions lead to multiple products with different redox states that possess a single hydroxyl group at C-2, a 2,3-vicinal diol, a contracted five-membered A-ring, or an expanded seven-membered A-ring. Similar reactions also take place in both natural products and other organic compounds harboring an epoxide adjacent to a carbonyl group that is conjugated to an aromatic moiety. Our findings extend the repertoire of known flavin chemistry that may provide new and useful tools for organic synthesis.

benzofluorene core of the kinamycins and lomaiviticins is further decorated with a diazo group endowing these compounds with potent antitumor activities that has attracted considerable attention 29,30 . The atypical angucyclines are often characterized by a highly oxygenated A-ring that can undergo further modifications such as acylation, epoxidation, glycosylation, and dimerization leading to significant structural diversity 18,21,27,31,32 . The α/β hydroxylases Alp1U and Lom6 were recently reported to catalyze stereoselective epoxide hydrolysis reactions during the biosynthesis of kinamycins and lomaiviticin 23 . Alp1U has also been shown to hydrolyze the epoxide of FST C (1) to afford the two stereoisomeric FSTs C1 (2) and C2 (3) (Fig. 1c) 18,33 .
The α/β-hydrolase FlsH from the FST pathway in the South China Sea-derived Micromonospora rosaria SCSIO N160 has been shown to catalyze deacylation of acyl FSTs; 18 however, it is unable to catalyze hydrolysis of the epoxide in FSTs despite its homology to Alp1U 18 . Naturally isolated FSTs ( Supplementary Fig. 1), such as FST B 12 , pyrazolofluostatins A-C 16 , and FST R 17 have been proposed to derive from the corresponding epoxide precursors; however, given the activity of FlsH it is not entirely clear how the epoxide would be cleaved and what enzymes if any would be responsible.
In this work, the 2,3-epoxide ring in FST C (1) is shown to open nonenzymatically in the presence of flavin adenine dinucleotide (FAD, 4) and nicotinamide adenine dinucleotide (NADH) with reduced FADH 2 (5) or C4a-hydroperoxyflavin (FADOOH, 6) serving as the reactive flavin species (Fig. 1c). The epoxide ring-opening reactions can thus proceed in a reductive manner to generate a putative enol intermediate, which is tautomerized to products possessing a single hydroxyl group at C-2 (7 and 8), or is further modified by 6 to products with a vicinal diol at C-2 and C-3 (3 and 9). In the presence of molecular oxygen, the epoxide ring opening reactions can also proceed in an oxidative manner leading to a peroxylated intermediate, which can be further reduced by NADH to 3 and 9, or can undergo ring rearrangements to afford products with a contracted five-membered A-ring (10) or a bridged and expanded sevenmembered A-ring (11). As a result, this non-enzymatic epoxide cleavage reaction may account for the formation of several different FST derivatives observed in Nature. Additional experiments show that similar reactions also take place in both natural products and other organic compounds harboring an epoxide adjacent to a carbonyl group that is conjugated to an aromatic moiety. Thus, this nonenzyme catalyzed ring-opening reaction holds promise as a useful tool in organic synthesis.

Results
Nonenzymatic epoxide ring-opening reaction of FST C (1) The α/β hydroxylase Alp1U was previously shown to hydrolyze the epoxide of FST C (1) to afford FST C1 (2) and FST C2 (3) (Fig. 2a, traces i-ii) 18,33 . In contrast, the Alp1U homologue FlsH is unable to catalyze hydrolysis of the epoxide ring in 1 (Fig. 2a, trace iii) 18 . A possible explanation is that FlsH might require a protein partner or an exogenous cofactor to catalyze the epoxide hydrolysis. In order to test these hypotheses, several flavin reductases including Fre from E. coli 34 were selected to examine their competence as putative protein partners for FlsH. It was found that incubation of FST C (1) with FlsH and Fre in the presence of NADH led to multiple products (Fig. 2a, trace vi). However, further experiments showed that formation of the same set of products was also observed in the absence of FlsH (Fig. 2a, trace vii), suggesting that Fre alone was able to mediate the same decomposition of FST C (1). Moreover, incubation of 1 with several other flavoenzymes including FlsO2 (prejadomycin oxidase) 35 , TiaM (tiacumicin halogenase) 36,37 , and XiaK (xiamycin N-hydroxylase) 38 also led to the same result (Supplementary Fig. 2). Given that the activities of these flavoenzymes all rely on the reduction of FAD by NADH, the reduced flavin alone was proposed to serve as the actual catalyst of FST C (1) consumption. Indeed, similar products were observed upon incubation of FST C (1) with FAD and NADH in the absence of any enzymes, such that FAD and NADH are both sufficient (Fig. 2a, traces viii & xii) and necessary (Fig. 2a, traces ix-xi) to facilitate the nonenzymatic decomposition of FST C (1).

Identification of the FST C decomposition products
Reaction products (3 and 7-11) were isolated from a scaled up reaction of FST C (1) with FAD and NADH and structurally characterized as shown in Fig. 1c. The first product was identified to be FST C2 (3) (Supplementary Fig. 3 and Table 1), which is also a product of the Alp1U-catalyzed hydrolysis reaction 33 . The second reaction product 7 (Supplementary Fig. 4 and Table 2) was established to have the same planar structure as FST B, an FST naturally isolated from Streptomyces sp. TA-3391 12 and Streptomyces strain Acta 1383; 13 however, the absolute configuration of FST B was not determined 12 . The (1R,2R,3S) absolute configuration of 7 was established by the solution TDDFT-ECD methodology ( Supplementary Fig. 5) and confirmed by single crystal X-ray analysis (Fig. 2b, CCDC 2036399; Supplementary Table 3). The third product 8 (Supplementary Fig. 6 and Table 2) was determined to have the same planar structure as 7 and the (1R,2R,3R) absolute configuration of 8 was assigned by comparison of the experimental and calculated ECD spectra carried out on the (1R,2R,3R) stereoisomer ( Supplementary Fig. 7) 39 . In order to distinguish the two stereoisomers of FST B, 7 and 8 were named FST B1 and FST B2 (Fig. 1), respectively. The product 9 (designated FST C3, Fig. 1) was isolated with the same retention time as that of 3 during in the HPLC analysis with a reversed-phase C18 column. Analysis of the assay using a polar HPLC column allowed separation of these two species (Fig. 2a, trace xii). Finally, 9 was identified to be a stereoisomer of 3 by HRESIMS and NMR data (Supplementary Fig. 8 and Table 4), and the (1R,2S,3S) absolute configuration of 9 was established by TDDFT-ECD methodology (Fig. 2c, Supplementary Fig. 9).
Two additional products 10 and 11 were also identified and structurally characterized ( Fig.1c; Fig. 2a, traces vi-viii & xii). The product 10, designated FST C4 (Fig. 1), was found to possess a contracted five-membered A-ring by NMR spectroscopic analysis (Supplementary Fig. 10 and Table 5) and was determined to have the (1R,2R) absolute configuration by single crystal X-ray analysis ( Fig. 2b; Supplementary Table 6; CCDC 2129081). The product 11, designated FST C5 (Fig. 1), was structurally elucidated to have a bridged and expanded sevenmembered A-ring (Supplementary Fig. 11 and Table 5). Assignment of the (1R,2R,3R) absolute configuration to 11 was based on its origin from 1 but otherwise remains tentative, because a definitive experimental characterization was not possible on account of its instability. Currently, there are no reports of 9-11 having been isolated from a natural source. 5 1 0  Labeling studies of the epoxide ring-opening reaction To investigate the mechanism of the nonenzymatic epoxide ring opening reaction, FST C (1), FAD and NADH were coincubated in PBS buffer (pH 7.0) prepared with deuterium oxide ( 2 H 2 O). Incorporation of a single deuteron into both 7 and 8 was indicated by the presence of a + 1 Da-shifted molecular ion peak at m/z 326.7 ([M -H] -) for both reduced products ( Supplementary Fig. 12). In contrast, no changes in the molecular weights of 3, 9, 10 or 11 ( Supplementary Fig. 12) were observed. Subsequently, 7-2 H and 8-2 H were isolated from scaled up reactions and analyzed by 1 H and 13 C NMR (Supplementary Figs. 13, 14 and Table 2). Comparison of the 1 H NMR spectra of 7-2 H and 7 revealed that the 3-H proton signal of 7 had vanished in 7-2 H and meanwhile the 3-Me doublet of 7 had collapsed to a singlet in 7-2 H (Fig. 3a), which located 2 H at C-3 of 7-2 H. In support of this assignment, the COSY correlation between the H-2 and H-3 protons observed in 7 was absent in 7-2 H ( Supplementary Figs. 4, 13). Likewise, a singlet was also observed for the 3-Me along with the absence of any COSY correlation between H-2 and H-3 in 8-2 H ( Fig. 3a; Supplementary Figs. 13,14). Moreover, the 13 C NMR spectra of 7-2 H and 8-2 H both showed broadening and reduced intensity of the signals from C-3, and 2 H-13 C couplings at C-3 were also observed (Supplementary Figs. 13,14) 40 . These observations demonstrate that a single solvent hydron is incorporated at C-3 during the reduction of 1 to both 7 and 8.
When FST C (1) was incubated with NADH and FAD under 18 Fig. 15). These data indicate that the conversion of 1 to 3, 9, 10, and 11 involves incorporation of one oxygen atom from O 2 . Furthermore, the relative production of 3/9 and 10/11 versus 7/8 was O 2 dependent, because incubation of FST C (1) with FAD and NADH after saturating the reaction buffer with O 2 led to a significant increase in the levels of the former (3,(9)(10)(11) versus the latter (7 and 8); in contrast, saturating the reaction buffer with N 2 led to 7 and 8 as the dominant products (Fig. 3b, traces iii and iv).  Fig. 17). Specifically, comparison of the 13 C NMR data for 9 and its 18 O isotopolog 9-18 O demonstrated a 3.8 Hz upfield shift of δ C-3 in the latter relative to that of 9 (Supplementary Fig. 18   Probing parameters affecting the epoxide ring opening reactions The pH dependence of the FAD/NADH-dependent decomposition of 1 was subsequently investigated by performing the reaction in buffers over the pH range 3-10. Following the incubation of 100 μM 1 with 100 μM FAD and 10 mM NADH for 2 h at 30°C, 1 was more efficiently converted to the epoxide ring-opened products with buffers in the pH range 5-7; however, the reaction was less efficient under more alkaline conditions at pHs 9-10 ( Supplementary Fig. 19). FST B1 (7) was rather stable at pH 3-7; however, it readily converted to 8 at higher pH ( Supplementary Fig. 20), where it also underwent oxidation to yield the previously reported natural product FST A (12) (Fig. 3b, trace v; Supplementary Fig. 20 and Table 1) Fig. 23). Based on these measurements, the intermediate is tentatively proposed to be the enol 13 (Fig. 3b), which is essentially a tautomer of 7 and 8 and thus has the same oxidation state as these two products.
While several attempts were made to isolate the intermediate in order to confirm its structural assignment as 13, only 7 was obtained as the major product along with another minor compound 14. This is consistent with the poor stability of the intermediate and its facile conversion to 7 (Fig. 3b, trace vii, Supplementary Fig. 24). Nevertheless, 14 could be characterized and was thus identified as the C-3 peroxide designated hydroperoxyfluostatin (  Table 5); however, poor recovery of 14 hampered collection of sufficient spectra for a complete characterization of its absolute configuration at C-3. Hydroperoxyfluostatin (14) was stable in water, even in the presence of FAD (Fig. 3b, traces x and xi). While the coincubation of 14 with NADH alone led to multiple products 3, 9, 10 and 11, the inclusion of FAD with NADH did not change the product profile ( Fig. 3b, traces xii and xiii; Supplementary Fig. 26). Given the observed conversion of 14 to both 3 and 9, compound 14 is likely a mixture of the (1S,2S,3R) and (1S,2S,3S) stereoisomers. The fact that isolation of 13 afforded a minor amount of 14 suggests that 14 is another intermediate that coelutes with 13 as opposed to being derived from 13. Furthermore, while 3/9 could be generated from both 13 and 14, 7/8 were only produced from 13, whereas 10/11 were exclusively derived from 14 (Fig. 3a).
When other flavin-related cofactors were considered, FMN and riboflavin in place of FAD were also found to efficiently mediate the decomposition of 1 in the presence of NADH (Fig. 3b, traces xiv-xvi). Isoalloxazine and NADH were also able to facilitate the formation of 3 from 1, albeit with a much lower efficiency (Fig. 3b, trace xvii). Only the products 3, 9-11 were observed using the cofactor F 420 in the presence of the reducing system constructed from glucose 6-phosphate and F 420 -dependent glucose-6-phosphate dehydrogenase (FGD) [43][44][45] , but notably no production of the reduced species 7 and 8 was observed (Fig. 3b, trace xviii). The observation of oxygen incorporation in the presence of F 420 and NADH was unexpected, because reduced 5-deazaflavin has generally be reported to react very slowly with molecular oxygen 46,47 . Nevertheless, the results with flavin cofactor compatibility shown in Fig. 3b (traces xiv-xviii) were found to be reproducible ( Supplementary Fig. 27). NADPH was also found to be an effective cofactor similar to NADH and thus support the epoxide ring opening reactions (Supplementary Fig. 19).
Two common structural features shared by all compounds that can react with FAD/NADH to undergo the epoxide ring opening reactions include (i) the presence of a carbonyl adjacent to the epoxide and (ii) the presence of an aromatic moiety adjacent to the carbonyl/ epoxide pair (Fig. 4). The absence of a carbonyl group neighboring the epoxide ring in 21 and 28-31 may account for the observed lack of reactivity. While 36 and 37 underwent ring opening in the presence of FAD/NADH, no reaction was observed for 32-35 despite the presence of an epoxide/carbonyl pair in these compounds. This implies that conjugation of the carbonyl/epoxide pair to an aromatic system is also necessary for ring opening of the epoxide.
The commercial compound vitamin K1 2,3-epoxide (42) was also tested as a putative substrate, which features both an epoxide/carbonyl pair and an adjacent aromatic ring. However, incubation of 42 with FAD/NADH led to no changes in either its TLC retention profile or spectroscopic properties implying no reaction (Supplementary   Fig. 54). A recent study demonstrated that the flavoenzyme RslO5 catalyzed a hydride-mediated reductive epoxide ring opening reaction to convert 43 to 44 during rishirilide biosynthesis (Fig. 4) 11 . The reaction proceeded in a manner that was highly similar to the conversion of 1 to 7. However, the production of 44 from 43 was confirmed to be strictly RslO5-dependent, and 43 was unreactive in the presence of FMN and NADH 11 . A possible explanation is that the presence of a long side chain next to the epoxide in both 42 and 43 might hinder both approach and orientation of the flavin thereby preventing suitable interactions for ring opening (Supplementary Fig. 55). Two additional commercial compounds trans-1,3-diphenyl-2,3-epoxypropan-1-one (45) and chalcone α,β-epoxide (46) were found to be unreactive with FAD/NADH; however, 46 but not 45 was accepted as a substrate by RlsO5 to yield the single product 47 (Supplementary Table 13 Supplementary Fig. 58) 52 . Previous studies demonstrated that the RslO5-H172N mutant had a deficiency in FMN binding and was inactive with the native substrate 43 11 , suggesting the critical role of FMN in the RslO5-catalyzed reaction. Taken together, a hydride from FMNH 2 originating from NADH is proposed to be the effective nucleophile for RslO5-catalyzed epoxide opening reactions. In contrast, no deuterium incorporation into 7 was observed in reactions of 1 and FAD with either (S)-[4-2 H]NADH or (R)-[4-2 H]NADH generated as described above (Supplementary Figs. 58, 59). Therefore, nonenzymatic reduction of 1 to 7 in the presence of FAD/NADH differs from that of RslO5 catalysis with respect to the ultimate origin of the C-3 hydrogen.

Discussion
Epoxide ring opening reactions are common and important in both biological processes and synthetic applications 1,2 . Nonenzymatic epoxide ring opening reactions are typically redox neutral proceeding through nucleophilic addition with the preferred regioselectivity and stereoselectivity contingent upon the nature of the epoxide and on the reaction conditions to generally yield products with a trans-vicinal diol 3,4 . Similar chemistry is encountered among the α/β-epoxide hydrolases that also generate trans-vicinal diol products 53 . For example, the hydrolase AlpU catalyzes the epoxide hydrolysis of the FST C (1) epoxide to afford 2 and 3, each of which exhibits a transvicinal diol (Fig. 1) 33 . In contrast, the flavoenzyme RslO5 was recently reported to catalyze a reductive epoxide ring opening of rishirilide thereby yielding a monohydroxylated product 11 . The RslO5 chemistry is thus distinctly different from that of the previously reported hydrolytic reactions and instead represents hydrogenation of an epoxide by reduced flavin, which is evident from the results using isotopically labeled substrates (Supplementary Fig. 58).
In this study, FAD/NADH is shown to induce the ring opening of suitably activated epoxides leading to a variety of products with different redox states. Under anaerobic conditions epoxide ring opening proceeds reductively in the presence of FAD/NADH via a mechanism that may be unrelated to that of RslO5 catalysis. The resulting monohydroxylated species, however, are highly susceptible to oxidation by molecular oxygen resulting in quinones (e.g., 12) and vicinal alcohols in reactions that can also be accelerated by the presence of FAD. Under aerobic conditions, the epoxide ring opening appears to proceed through the formation of an α-ketoperoxylated species which can be reduced by NADH to again generate vicinal diols. Alternatively, the peroxylated intermediate can undergo further rearrangement leading to both ring contracted (e.g., the five-membered species 10) as well as ring expanded (e.g., the seven-membered species 11) oxidized products.
On the basis of the accumulated experimental evidence, a putative mechanism is proposed for the FAD/NADH enabled nonenzymatic epoxide-ring opening reactions (Fig. 5). Since both FAD and excess NADH are required for the observed epoxide ring opening reactions, NADH is proposed as the reductant with FAD serving as a redox catalyst. When the reaction is conducted aerobically, O 2 is proposed to act as the oxidant. Given that NADH alone could not initiate the reaction, the reduced flavin is proposed to be the species directly responsible for the reduction of 1. It is possible that 7 is directly formed via a hydride transfer from a reduced flavin species (e.g., N5-H of FADH 2 ) to C3 of 1 thereby opening the epoxide ring, which would resemble the RslO5 catalyzed epoxide reduction reaction (Fig. 4). In this case, the observation that the product 7 has its H-3 derived from solvent could be explained by ready exchange of N5-H of the reduced flavin with solvent. This would imply that 7 should be the immediate reduction product prior to tautomerization to the observed intermediate 13.
However, a time course assay using 0.5 μM 1 with 10 μM FAD and 2 mM NADH demonstrated that 13 accumulates first followed by conversion to 7 and 8. In contrast, no significant accumulation of 13 was observed in incubations of 7 under similar conditions ( Supplementary Fig. 60). These results imply that 13 is the initial reduction product rather than 7 or 8, and thus nonenzymatic reduction of 1 in the presence of FAD/ NADH does not proceed via direct hydride transfer to C-3 as is proposed for RslO5 catalysis.
An alternative mechanism involves activation of the oxirane by the adjacent carbonyl coupled to an aromatic ring and undergoes reduction leading to an unstable intermediate. This intermediate is unlikely to be 7-demethyl-FST Q (48) (Fig. 4), which is the product of a direct C-4 carbonyl reduction in 1, because the previously isolated FST Q (21) 17 is inert to the FAD/NADH reaction system (Supplementary Fig. 40). Instead, the intermediate during reduction of FST C (1) is hypothesized to be an enol species such as 13, which is consistent with the observation that 13 readily tautomerizes to yield 7 and 8. This mechanism is also consistent with the incorporation of deuterium at H-3 of 7 and 8 when the reaction is carried out in 2 H 2 O. Compound 13 is also expected to be susceptible to autoxidation to yield 3 and 9 in a manner that is sensitive to the presence of O 2 , FAD, and NADH.
Formation of 13 may proceed via the addition of the nucleophilic N-5 of the reduced flavin to 1. This model is supported by the fact that no formation of 7 was observed using the reduced F 420 cofactor, which lacks the nucleophilic N-5 but is still able to serve as a hydride donor 54 . Nucleophilic addition of FADH 2 to 1 may occur at either C-3 or C-4. Given the presence of a sterically bulky CH 3 group at C-3 of 1, C-4 is proposed to be the likely site of nucleophilic attack, which is consistent with the requirement for a carbonyl adjacent to the epoxide and thus the different reactivity of 16 (C-4 carbonyl) versus 21 (C-4 hydroxyl). Considering that N-5 in FADH 2 possesses a lone pair, and C-4 in 1 is a trigonal carbonyl carbon, the redox reaction is proposed to happen via an n→π* interaction. Specifically, the nucleophilic N-5 donates the lonepair (n) electron density into the trigonal carbene center (empty π* orbital) along the Burgi-Dunitz trajectory 55 . Mixing of these orbitals is thermodynamically favorable resulting in a covalent adduct 49 (Fig. 5) 56 . Subsequent N-1 deprotonation of the flavin moiety in 49 leads to the breakage of the N 5 -C 4' linkage and release of the oxidized flavin thereby completing the reduction of 1 to the enol intermediate 13 with a Δ 3,4 double bond (Fig. 5).
Intermediate 13 is highly unstable and undergoes facile tautomerization to incorporate a solvent hydron at the α-carbon (C-3) either from the si face (the major route) or the re face (the minor route) of the sp 2 planar structure, to yield 7 or 8, respectively 57 . This mechanism is consistent with the site-specific incorporation of deuterium at C-3 in 7 and 8 from buffered 2 H 2 O (Fig. 3a). Despite the 2,3-cis configuration of 7 compared to 8, the former appears to be the thermodynamically favored tautomer with an equilibrium constant of approximately [7] eq / [8] eq = 5.4 ( Supplementary Fig. 61). Moreover, 7, 8 and the putative enol intermediate 13 are also susceptible to autooxidation to yield 12 ( Supplementary Fig. 62).
The putative enol intermediate 13 also appears to be sensitive to molecular oxygen and can undergo autoxidation in the presence of FAD and NADH. Reduced flavin is known to react with O 2 to form the covalent Fl 4aOOH adduct (FAD → FADOOH) 58 , and in some cases, H 2 O 2 may be released from FADOOH to serve as the oxidant 37,59 . However, neither 1 nor 13 can react with H 2 O 2 ( Supplementary Figs. 24, 63). Thus, the intermediate 13 is proposed to react with the electrophilic C(4a)hydroperoxy group of FADOOH to afford the C-3 monooxygenated products (see Supplementary Fig. 64). Hydroxylation can occur either from the si face (major route) or the re face (minor route) of the planar double bond (Δ 3,4 ) to produce 3 or 9, respectively, with concomitant release of the oxidized flavin (Fig. 5). The proposed mechanism is supported by the observations that (i) the O incorporated into both 3 and 9 at C-3 originates in O 2 , and (ii) the conversion of 13 to 3 and 9 requires the presence of FAD, NADH and O 2 .
Isolation of intermediate 14 indicates that 3 and 9 can also be produced in an alternative way that does not involve 13. In the presence of molecular oxygen, the flavin C(4a)-peroxide (FADOO -) may serve as a nucleophile to regioselectively attack C-3 of 1 at either the si face (major route) or the re face (minor route) to open the epoxide ring, resulting in the putative adduct 50 (Fig. 5). The presence of the C-4 carbonyl oxygen with electron-withdrawing effect may cause changes of the electron density of C-3 60 , to make C-3 a better site for nucleophile substitutions than C-2 of the epoxide in 1. Deprotonation of the flavin N-5 in 50 breaks the C 4a -O covalent bond to release 14 and FAD (Fig. 5). The product 14 is a mixture of C-3 isomers and easily reduced to 3 and 9 by NADH (Fig. 5, Supplementary Fig. 65). Coincubation of 14 with NADH also leads to 10 and 11, which is proposed to involve a Baeyer-Villiger reaction-like mechanism. The C-3 hydroperoxide in 14 may thus perform an intramolecular nucleophilic addition at the C-4 carbonyl to form the putative intermediate 51, which can partition to generate 10 and 11. In the former case, intermediate 51 may undergo a Criegee rearrangement to yield a seven-membered lactone ring 61,62 , followed by an intramolecular backbiting addition of the C-1 hydroxy at the C-4 carbonyl that leads to 10 after dehydration (Fig. 5) 63 . Alternatively, the 1,2-dioxetane unit of 51 may undergo heterolytic cleavage, followed by Grob-like fragmentation to yield an oxoheptanoate intermediate 64,65 , which eventually generates the hemiacetal 11 after successive intramolecular cyclizations and dehydration 66 .
In conclusion, the reductive epoxide ring opening is suggested to proceed mechanistically via a transient adduct (49) with an N 5 -C 4' , covalent bond that is formed between N-5 of the flavin cofactor and C-4 of the substrate FST C (1) (Fig. 5). While tautomerization of the resulting product can yield 7 and 8, nucleophilic reaction with FADOOH can also afford the diol products 3 and 9. Oxidative epoxide ring opening of 1 produces the intermediate 14 via a putative C 4a -O-O-C 3' covalent adduct (50, Fig. 5). The canonical reactivity of flavin cofactors involves redox chemistry through N-5 hydride transfer or oxygen activation at C-4a in the majority of cases 67 . However, a few flavoenzymes have been reported to catalyze the formation of covalent flavin N-5-substrate adducts in a redox-neutral manner 68 , with examples including the iminium-adducts in the catalytic cycle of UDP-galactopyranose mutase 69 , alkyl-dihydroxyacetone phosphate synthase 70 , flavin prenyltransferase 71 , thymidylate synthase 72 , and 2-haloacrylate hydratase 73 .
The present study provides an example likely involving flavin N-5 adduct formation during flavin-catalyzed nonenzymatic reactions (Fig. 5). Furthermore, in addition to serving as a nucleophile in mediating hydroxylation, epoxidation and Baeyer-Villiger oxidation in flavoenzyme catalysis 58 , the flavin C(4a)-peroxide (FADOO -) is proposed to also facilitate the formation of peroxylated intermediates such as 14 (Fig. 5). Flavin and flavin derivatives are well known to participate as cofactors in a wide variety of reactions found in nature 67,74 . Previous reports have shown that natural flavins can catalyze nonenzymatic oxidative decarboxylation of picolinic acid derivatives 75 and the nonenzymatic iodination of diverse aromatic compounds 37,59 . In this study, the chemistry of flavins is expanded to epoxide ring opening reactions in both a reductive and an oxidative manner to generate multiple ring-opened products. Consequently, the flavin chemistry identified in this study may be applicable to other epoxides and thus holds promise as a useful tool in organic synthesis.

Bacterial strains, compounds, and reagents
The strain Micromonospora rosaria SCSIO N160 was used for the isolation of FSTs 15 . FST Q (21) was previously isolated from Streptomyces sp. PKU-MA00045 17 . Chemicals, enzymes, and other molecular biological reagents were purchased from standard commercial sources and used according to the recommendations of the manufacturer.

Expression, purification of recombinant enzymes, and general in vitro enzyme assays
The DNA fragment of rslO5 (GenBank: KJ437438.1 [https://www.ncbi. nlm.nih.gov/nuccore/KJ437438.1]) from the rishirilide biosynthetic gene cluster in Streptomyces bottropensis 11 was synthesized and cloned into pET28a to afford the plasmid pCSG8112 (Supplementary  Table S14). When the cultures of E. coli BL21(DE3)/pCSG8112 in LB media containing kanamycin (50 μg mL −1 ) were grown to an OD 600 of about 0.6 at 37°C, the production of RslO5 was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM. The cultures were grown at 16°C for an additional 12 h. The cells were then collected by centrifugation and were resuspended in the lysis buffer (20 mM Tris-Cl, 500 mM NaCl, and 5 mM imidazole, pH 8.0) for sonication. Purification of N-His 6 -tagged RslO5 was conducted using Ni-NTA affinity chromatography according to the manufacturer's manual (Novagen, USA). After desalting with PD-10 column (GE Healthcare, USA), the purified RslO5 was stored in the storage buffer (10% glycerol, 1 mM DTT, 50 mM Tris-Cl, 100 mM NaCl, pH 8.0) at −80°C for further use. The expression and purification of Fre 34 , Alp1U 18 , FlsH 18 , XiaK 38 , TiaM 36 , and FlsO2 35 were performed similar as described for RslO5. The standard in vitro enzyme assays (Alp1U, FlsH, Fre, XiaK, TiaM, Fre, or FlsO2) contained 100 μM FST C (1) and 10 μM of purified recombinant enzyme in 50 mM PBS (phosphate buffered saline) buffer (pH 7.0) in a total volume of 100 μL with incubation at 30°C for 0.5-2 h. The reactions were stopped by adding 100 μL of ice-cold MeOH and were monitored using the general analytical HPLC methods.

Nonenzymatic reactions with flavins
A typical reaction mixture included 100 µM 1, 100 µM FAD and 10 mM NADH in 50 mM PBS buffer (pH 7.0) in a total volume 100 µL with incubation at 30°C for 0.5-2 h. In general, the reactions were terminated by adding 100 μL of ice-cold MeOH and analyzed by HPLC using the general analytical HPLC method or subjected to LC-MS analysis. For determining the pH effects on the reactions, the assays were performed in a total volume of 100 μL containing 100 μM 1, 100 μM FAD and 10 mM NADH in 50 mM buffers with pH values ranging from 3 to 10 with incubation at 30°C for 2 h. The buffers were prepared as follows: citric acid/Na 2 HPO 4 buffer (pH 3-6); PBS buffer (pH 7); boric acid/borax buffer (pH 8-9), borax/NaOH buffer (pH 10). As controls, 100 μM 1 was incubated in different pH buffers (pH 3-10) in the presence of 100 μM FAD alone (or 10 mM NADH alone) at 30°C for 2 h.
To test the reactivity of H 2 O 2 with FST C (1), the assays were performed in a total volume of 100 μL containing 100 μM 1 and 30% H 2 O 2 in 50 mM buffers at pH 3.0 (or pH 7.0 or pH 10.0) by incubation at 30°C for 2 h.

The analysis of the intermediate 13
A reaction mixture containing 100 µM 1, 10 µM FAD, and 2 mM NADH in 50 mM PBS buffer (pH 7.0) in a total volume 100 µL was incubated under a normal atmosphere at 30°C for 20 min. The reactions were quenched by adding 100 μL of ice-cold MeOH. To check the stability of 13, the intermediate 13 was collected from a general analytical HPLC run and was immediately reinjected for HPLC analysis. Similarly, to monitor the changes of 13 under various conditions, the freshly collected 13 was immediately incubated in 50 mM PBS buffer (pH 7.0) containing (i) 10 µM FAD; (ii) 30% H 2 O 2 ; (iii) 2 mM NADH; (iv) 10 µM FAD and 2 mM NADH (in presence of excess of O 2 ) at 30°C for 30 min. The reactions were quenched by adding 100 μL of ice cold MeOH and monitored by the general analytical HPLC method using a C18 column as well as a polar column.

Isolation and stability of 14
Scaled up reactions in a total volume of 50 mL (100 µL × 500 Eppendorf tubes) were performed containing 100 µM 1, 10 µM FAD, and 2 mM NADH in 50 mM PBS buffer (pH 7.0) at 30°C for 20 min with the aim of isolating 13 for structure elucidation. The reactions in each Eppendorf tube were stopped by extracting three times with 100 μL of ice-cold butanone and the solvents were removed under vacuum on an ice bath. The crude extracts were then collected and dissolved in 1.0 mL MeOH and subjected to general analytical HPLC using a polar column. The collections were kept at −80°C prior to lyophilization. The collected fractions were purified a second time to remove 7, which had been generated from the decomposition of 13. This process finally afforded a pure and stable compound (0.8 mg), which was dissolved in DMSO-d 6 for NMR analysis and confirmed to be 14.
A set of assays were performed to check the stability of 14. A typical reaction mixture included 100 µM 14, 10 µM FAD, and 2 mM NADH in 50 mM PBS buffer (pH 7.0) in a total volume 100 µL. Control reactions were run in parallel by omitting FAD or NADH in the assays. Co-incubation of 100 µM 14 in 30% H 2 O 2 were also performed in a total volume of 100 μL. The reaction mixtures were incubated at 30°C for 30 min. The reactions were quenched after adding 100 μL of ice-cold MeOH and were analyzed by HPLC using a polar column following the general analytical HPLC method.
In vitro RslO5 assays, isolation of 47 and deuterium incorporation studies A standard RslO5 in vitro reaction mixture contained 100 μM trans-1,3diphenyl-2,3-epoxypropan-1-one (45) or chalcone α,β-epoxide (46), 10 μM RslO5 and 5 mM NADH in 50 mM PBS buffer (pH 7.0) in a total volume of 100 μL and incubated at 30°C for 2 h. The reaction with 46 was also performed in 50 mM PBS buffer (pH 7.0) prepared with deuterated water ( 2 H 2 O) and subjected to LC-HRESIMS analysis to investigate 2 H incorporation into 47 from the solvent. To isolate 47 for structural elucidation, 15 mL reactions were performed in 50 mM PBS buffer (pH 7.0) containing 100 μM 46, 10 μM RslO5 and 5 mM NADH at 30°C for 6 h. The reaction was quenched with an equal volume of prechilled butanone. After centrifugation at 2057 × g for 20 min at 4°C, the supernatants were extracted three times with an equal volume of pre-chilled butanone. The butanone was removed under vacuum on an ice bath. The crude extracts were dissolved in 500 μL of MeOH and were purified by semi-preparative HPLC using a C18 column (250 mm × 10.0 mm, 5 μm; Phenomenex, USA), with an isocratic elution gradient of 60% A (H 2 O with 0.8% formic acid)/40% B (MeCN) at a flow rate of 2.5 mL min −1 to yield 47 (3.0 mg, 41.0%).
The purification of D-glucose dehydrogenases BmGDH from Bacillus megaterium DSM 2894 in E. coli BL21(DE3)/pRSF-BmGDH and TaGDH from Thermoplasma acidophilum in E. coli BL21(DE3)/ pCSG3622, was carried out according to our previous study 76 . In case of the RslO5/GDH coupling assays, the reduction of NADP to NADPH was first performed in a 100 μL reaction containing 10 μM TaGDH (or BmGDH), 2 mM NADP and 100 mM [1-2 H]D-glucose in 50 mM sodium phosphate buffer (pH 7.0) by incubation for 1 h at 37°C for BmGDH or 50°C for TaGDH, followed by the addition of 100 μM 46 and 10 mM RslO5 and further incubation at 30°C for 6 h. The in situ generated 2 H-labeled NADH was also reacted with 1 and FAD to monitor the incorporation of 2 H in to the product 7. The reactions were quenched with an equal volume of ice-cold MeOH and analyzed by LC-HRESIMS using the general analytical HPLC method.

Quantum chemistry calculations
Mixed torsional/low-mode conformational searches were carried out by means of the Macromodel 10.8.011 software using the MMFF with an implicit solvent model for CHCl 3 applying a 21 kJ/mol energy window 77 . Geometry re-optimizations of the resultant conformers were carried out at the ωB97X/TZVP level with the PCM solvent model for MeCN. TDDFT ECD calculations were performed with Gaussian 09 for ECD using various functionals (B3LYP, BH&HLYP, CAM-B3LYP, PBE0) and the TZVP basis set with the same solvent model as in the preceding DFT optimization step 78 . ECD spectra were generated as the sum of Gaussians with 3000 and 3600 cm −1 half-height widths, using dipole-velocity-computed rotational strength values 79 . Boltzmann distributions were estimated from the ωB97X energies. The MOLEKEL software package was used for visualization of the results 80 .

X-ray crystallographic analysis
A single crystal of 7, 10, and 23 was obtained in a mixed solvent of MeOH and water, and single crystals of 40 were obtained in a mixed solvent of CH 3 CN and H 2 O. The suitable crystals were selected, and the crystal data were recorded on an XtaLAB AFC12 (RINC): Kappa single diffractometer, with Cu Kα radiation (λ = 1.541 84 Å). The crystals were kept at 99.9(7) K for 7, 99.8(9) K for 23, 100.00(10) K for 10 and 100.01(12) K for 40 for during data collection. Using Olex2 81 , the structure was solved with the ShelXT 82 structure solution program using intrinsic phasing and refined with the ShelXL refinement package using least squares minimization.